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We hypothesized that inhibition of the cGMP-specific enzyme phosphodiesterase 5A (PDE5A) promoted cGMP/protein kinase G (PKG) activity to condition stem cells for enhanced survival and proliferation. One-time tadalafil treatment (1 [micro]M for 30 min) of mesenchymal stem cells ([sup.Tada]MSCs) provided sustained protection of cells for 36 h. Higher cGMP activity with concomitantly increased PKGI activity was observed in [sup.Tada]MSCs, which peaked within 12 h after tadalafil treatment. Pretreatment with PKG1 blockers (1 [micro]M KT-5823 or 20 nM K-252a) or transduction with adenoviral PKG1-short-hairpin RNA abolished tadalafil-induced cytoprotection of the cells. A higher proliferation rate was observed in 7adaMSCs compared with nontreated MSCs ([sup.Cont]MSCs). In a rat model of acute myocardial infarction, [sup.Tada]MSCs transplanted 0 and 24 h after tadalafil treatment showed higher survival compared with [sup.cont]MSCs on day 2 and day 4 after engraftment. [sup.Tada]MSCs transplanted 48 h after tadalafil treatment lost their protection on both day 2 and day 4 after engraftment, and their rate of survival was similar to [sup.cont]MSCs. Reduced terminal dUTP nick end-labeling positivity (P < 0.01 vs. [sup.Cont]MSCs) and higher proliferation of [sup.Tada]MSCs (P < 0.01 vs. [sup.Cont]MSCs) was observed in the infarcted heart. Fluorescence immunostaining revealed neomyogenesis in both the infarct and peri-infarct areas. Blood vessel density was significantly increased in group 2 compared with group 1. Transthoracic echocardiographic heart function revealed significant preservation of the indexes of left ventricle contractility and attenuation of remodeling in [sup.Tada]MSC-engrafted animal hearts (group 2) compared with [sup.Cont]MSCs (group 1). PDE5A inhibition using long-acting tadalafil is an innovative approach to promote stem cell survival and proliferation in the infarcted heart. cytoprotection; phosphodiesterase; preconditioning; stem cells; tadalafil doi: 10.1152/ajpheart.00437.2010.

The Escherichia coli K-12 yfgF gene encodes a protein with domains associated with cyclic di-GMP signalling: GGDEF (associated with diguanylate cyclase activity) and EAL (associated with cyclic di-GMP phosphodiesterase activity). Here, it is shown that yfgF is expressed under anaerobic conditions from a class II FNR (regulator of fumarate and nitrate reduction)-dependent promoter. Anaerobic expression of yfgF is greatest in stationary phase, and in cultures grown at 28[degrees]C, suggesting that low growth rates promote yfgF expression. Mutation of yfgF resulted in altered cell surface properties and enhanced sensitivity when anaerobic cultures were exposed to peroxides. The purified YfgF GGDEF-EAL ([YfgF.sub.GE]) and EAL ([YfgF.sub.E]) domains possessed cyclic di-GMP-specific phosphodiesterase activity, but lacked diguanylate cyclase activity. However, the catalytically inactive GGDEF domain was required for [YfgF.sub.GE] dimerization and enhanced cyclic di-GMP phosphodiesterase activity in the presence of physiological concentrations of [Mg.sup.2+]. The cyclic di-GMP phosphodiesterase activity of [YfgF.sub.GE] and [YfgF.sub.E] was inhibited by the product of the reaction, 5'-phosphoguanylyl-(3'-5')-guanosine (pGpG). Thus, it is shown that the yfgF gene encodes an anaerobic cyclic di-GMP phosphodiesterase that is involved in remodelling the cell surface of E. coli K-12 and in the response to peroxide shock, with implications for integrating three global regulatory networks, i.e. oxygen regulation, cyclic di-GMP signalling and the oxidative stress response. DOI 10.1099/mic.0.037887-0

Bacteria produce different types of biofilms under distinct environmental conditions. Vibrio fischeri has the capacity to produce at least two distinct types of biofilms, one that relies on the symbiosis polysaccharide Syp and another that depends upon cellulose. A key regulator of biofilm formation in bacteria is the intracellular signaling molecule cyclic diguanylate (c-di-GMP). In this study, we focused on a predicted c-di-GMP phosphodiesterase encoded by the gene binA, located directly downstream of syp, a cluster of 18 genes critical for biofilm formation and the initiation of symbiotic colonization of the squid Euprymna scolopes. Disruption or deletion of binA increased biofilm formation in culture and led to increased binding of Congo red and calcofluor, which are indicators of cellulose production. Using random transposon mutagenesis, we determined that the phenotypes of the [DELTA]binA mutant strain could be disrupted by insertions in genes in the bacterial cellulose biosynthesis cluster (bcs), suggesting that cellulose production is negatively regulated by BinA. Replacement of critical amino acids within the conserved EAL residues of the EAL domain disrupted BinA activity, and deletion of binA increased c-di-GMP levels in the cell. Together, these data support the hypotheses that BinA functions as a phosphodiesterase and that c-di-GMP activates cellulose biosynthesis. Finally, overexpression of the syp regulator sypG induced binA expression. Thus, this work reveals a mechanism by which V. fischeri inhibits cellulose-dependent biofilm formation and suggests that the production of two different polysaccharides may be coordinated through the action of the cellulose inhibitor BinA. doi: 10.1128/JB.01048-09

A study highlights that autosomal-dominant striatal degeneration (ADSD), a movement disorder affecting the striatal part of the basal ganglia, is caused by a complex frameshift mutation in the phosphodiesterase 8B (PDE8B) gene expressed in the brain. It shows that the mutation results in the loss of phosphodieterase enzyme activity, also suggesting that a functional analysis of PDE8B would help in a better understanding of ADSD and other such disorders.

Use of long-acting [[beta].sub.2]-adrenergic receptor ([beta]2AR) agonists to treat asthma incurs an increased risk of asthma morbidity with impaired bronchodilation and heightened bronchoconstriction, reflecting the adverse effects of prolonged homologous [beta]2AR desensitization on airway smooth muscle (ASM) function. Since phosphodiesterase 4 (PDE4) regulates ASM relaxation and contractility, we examined whether the changes in ASM function induced by prolonged homologous [beta]2AR desensitization are attributed to altered expression and action of PDE4. Cultured human ASM cells and isolated rabbit ASM tissues exposed for 24 h to the long-acting [beta]2AR agonist salmetero! exhibited impaired acute [beta]2AR-mediated cAMP accumulation and relaxation, respectively, together with ASM constrictor hyperresponsiveness. These proasthmatic-like changes in ASM function were associated with upregulated PDE4 activity due to enhanced expression of the PDE4D5 isoform and were prevented by pretreating the ASM preparations with the PDE4 inhibitor rolipram or with inhibitors of either PKA or ERK1/2 signaling. Extended studies using gene silencing and pharmacological approaches demonstrated that: 1) the mechanism underlying upregulated PDE4D5 expression following prolonged [beta]2AR agonist exposure involves PKA-dependent activation of [G.sub.i] protein signaling via its [beta][gamma]-subunits, which elicits downstream activation of ERKI/2 and its induction of PDE4D5 transcription; and 2) the induction of PDE4 activity and consequent changes in ASM responsiveness are prevented by pretreating the [beta]2AR agonist-exposed ASM preparations with inhibitors of [G.sub.i]-[beta][gamma] signaling. Collectively, these findings identify that the proasthmatic changes in ASM function resulting from prolonged homologous [beta]2AR desensitization are attributed to upregulated PDE4 expression induced by [G.sub.i]-[beta][gamma]-mediated cross-talk between the PKA and ERK1/2 signaling pathways. asthma; long-acting [[beta].sub.2]-agonists; salmeterol; cAMP signaling; G proteins; phosphodiesterase-4 doi: 10.1152/ajplung.00079.2009.

Previously, we demonstrated that genistein stimulated [Cl.sup.-] secretion in the mouse jejunum (Baker MJ and Hamilton KL, Am J Physiol Cell Physiol 287: C1636-C1645, 2004); however, the mode of action of genistein still remains unclear. Here, we examined the activation of [Cl.sup.-] secretion by the modulation of phosphodiesterases (PDEs) by genistein (75 [micro]M) in the mouse jejunum with the Ussing short-circuit current ([I.sub.sc]) technique. Drugs tested included theophylline (10 raM), a nonspecific PDE inhibitor; 8-methoxymethyl-3-isobutyl-l-methylxanthine (8-MM-IBMX; 100 [micro]M), erythro-9-(2-hydroxyl-3-nonyl)-adenine (EHNA; 40 [micro]M), milrinone (100 [micro]M), and rolipram (40 and 100 [micro]M), which are specific inhibitors of PDE1--PDE4, respectively. Theophylline stimulated a bumetanide-sensitive [I.sub.sc], indicative of [C1.sup.-] secretion, and abolished genistein's stimulatory action on [I.sub.sc]. Neither 8-MM-IBMX nor EHNA altered the basal [I.sub.sc] nor did these PDE inhibitors affect the stimulatory action of genistein on the [I.sub.sc] of the mouse jejunum. Rolipram had no effect on basal [I.sub.sc], but it reduced the genistein-stimulated [I.sub.sc] compared with time-matched control tissues. Milrinone stimulated a concentration-dependent increase in [I.sub.sc]. Bumetanide (10 [micro]M) inhibited 60 [+ or -] 4% of milrinone-induced [I.sub.sc]. Pretreating tissues with milrinone prevented genistein from stimulating [I.sub.sc], and pretreatment with genistein reduced the effect of milrinone on [I.sub.sc]. H89 (50 [micro]M), a PKA inhibitor, reduced the milrinone-stimulated [I.sub.sc]. Likewise, H89 reduced the genistein-stimulated [I.sub.sc]. Here, we demonstrate, for the first time, that genistein activates [Cl.sup.-] secretion of the mouse jejunum via inhibition of a PDE3-dependent pathway. PDE3; cystic fibrosis transmembrane conductance regulator; [Ca.sup.2+]activated [Cl.sup.-] channels; milrinone

The aims of this study were to study the influence of the duration of diabetes, the role of endothelial-derived vasodilators, and the role of phosphodiesterase (PDE) isoform activity in the early changes in vascular reactivity of aortic rings from diabetic rats. Diabetes mellitus was induced in female rats by intravenous streptozotocin (85 mg/kg). Two or 4 wk later, thoracic aortic rings from control and diabetic rats were isolated, and vascular responses to acetylcholine (ACh), S-nitrosoN-acetylpenicillamine (SNAP) [nitric oxide (NO) donor], DMPPO (PDE5 inhibitor), and phenylephrine (PE) were obtained in the presence and absence of endothelium or other drugs. PDE isoform activity was also measured. At 2 wk, responses to ACh and DMPPO were enhanced, whereas those to PE were attenuated in diabetic rats relative to controls. Indomethacin and SQ-29548 (a thromboxane [A.sub.2] receptor antagonist), but not [N.sup.G]-nitro-L-arginine methyl ester, corrected these differences. The responses to SNAP, and cAMP and cGMP hydrolytic activities, were similar in the two groups. In contrast, at 4 Wk, ACh, DMPPO, and PE produced similar responses in the two groups: [N.sup.G]-nitro-L-arginine methyl ester rendered the response to PE lower in the diabetic group, and this was corrected by indomethacin, but not SQ-29548, treatment. The response to SNAP was greater in the diabetic group, and this was corrected by DMPPO. Activity of all PDEs was decreased at 4 wk. We conclude that, at 2 wk, there is modulation of thromboxane [A.sub.2] production, but no change in the NO system or PDE isoform activities. At 4 wk, a reduction in NO activity is superimposed; at this stage, PDE activity is reduced, together with increased production of vasodilating prostaglandins, possibly as a compensatory mechanism to maintain normal vascular reactivity. endothelium; vasodilation; vasoconstriction

Glycerophosphodiesters are formed by deacylation of phospholipids. Streptomyces coelicolor and other soil-dwelling actinomycetes utilize glycerophosphodiesters as phosphate and carbon sources by the action of glycerophosphodiester phosphodiesterases (GDPDs). Seven genes encoding putative GDPDs occur in the S. coelicolor genome. Two of these genes, glpQ1 and glpQ2, encoding extracellular GDPDs, showed a PhoP-dependent upregulated profile in response to phosphate shiftdown. Expression studies using the luxAB genes as reporter confirmed the PhoP dependence of both glpQ1 and glpQ2. Footprinting analyses with pure GST-PhoP of the glpQ1 promoter revealed four protected direct repeat units (DRu). PhoP binding affinity to the glpQ2 promoter was lower and revealed a protected region containing five DRu. As expected for pho regulon genes, inorganic phosphate, and also glycerol 3-phosphate, inhibited the expression from both glpQ1 and glpQ2. The expression of glpQ1 was also repressed by serine and inositol but expression of glpQ2 was not. In contrast, glucose, fructose and glycerol increased expression of glpQ2 but not that of glpQ1. In summary, our results suggest an interaction of phosphate control mediated by PhoP and carbon source regulation of the glpQ1 and glpQ2 genes involving complex operator structures.

Fibrotic diseases are characterized by the accumulation of extracellular matrix together with distortion and disruption of tissue architecture. Phosphodiesterase (PDE)4 inhibitors, by preventing the breakdown of cAMP, can inhibit fibroblast functions and may be able to mitigate tissue remodeling. Transforming growth factor (TGF)-[beta]1, a mediator of fibrosis, can potentially modulate cAMP by altering [PGE.sub.2] metabolism. The present study assessed whether PDE4 inhibitors functionally antagonize the profibrotic activity of fibroblasts stimulated by TGF-[beta]1. The PDE4 inhibitors roflumilast and rolipram both inhibited fibroblast-mediated contraction of three-dimensional collagen gels and fibroblast chemotaxis toward fibronectin in the widely studied human fetal lung fibroblast strain HFL-1 and several strains of fibroblasts from adult human lung. Roflumilast was ~10-fold more potent than rolipram. There was a trend for PDE4 inhibitors to inhibit more in the presence of TGF-[beta]1 (0.05 < P < 0.08). The effect of the PDE4 inhibitors was mediated through camp-stimulated protein kinase A (PKA), although a PKA-independent effect on gel contraction was also observed. The effect of PDE4 inhibitors depended on fibroblast production of [PGE.sub.2] and TGF[beta]-induced [PGE.sub.2] production. PDE4 inhibitors together with TGF-[beta]1 resulted in augmented [PGE.sub.2] production together with increased expression of COX mRNA and protein. The present study supports the concept that PDE4 inhibitors may attenuate fibroblast activities that can lead to fibrosis and that PDE4 inhibitors may be particularly effective in the presence of TGF-[beta]1-induced fibroblast stimulation. phosphodiesterase 4; chemotaxis; collagen gel contraction; transforming growth factor-[beta]1; prostaglandin [E.sub.2]

Recent evidence indicates that 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), a marker enzyme of myelin and oligodendrocytes, is also present in neural and nonneural mitochondria. However, its role in mitochondria is still completely unclear. We found CNP in rat brain mitochondria and studied the effects of CNP substrates, 2',3'-cyclic nucleotides, on functional parameters of rat brain mitochondria. 2',3'-cAMP and 2',3'-cNADP stimulated [Ca.sup.2+] overload-induced [Ca.sup.2+] release from mitochondrial matrix. This [Ca.sup.2+] release under threshold [Ca.sup.2+] load correlated with membrane potential dissipation and mitochondrial swelling. The effects of 2',3'-cyclic nucleotides were suppressed by cyclosporin A, a potent inhibitor of permeability transition (PT). PT development is a key stage in initiation of apoptotic mitochondria-induced cell death. 2',3'-cAMP effects were observed on the functions of rat brain mitochondria only when PT was developed. This demonstrates involvement of 2',3'-cAMP in PT regulation in rat brain mitochondria. We also discovered that, under PT development, the specific enzymatic activity of CNP was reduced. Thus we hypothesize that suppression of CNP activity under threshold [Ca.sup.2+] load leads to elevation of 2',3'-cAMP levels that, in turn, promote PT development in rat brain mitochondria. Similar effects of 2',3'-cyclic nucleotides were observed in rat liver mitochondria. Involvement of CNP in PT regulation was confirmed in experiments using mitochondria from CNP-knockdown oligodendrocytes (OLN93 cells). CNP reduction in these mitochondria correlated with lowering the threshold for [Ca.sup.2+] overload-induced [Ca.sup.2+] release. Thus our results reveal a new function for CNP and 2',3'-cAMP in mitochondria, being a regulator/promotor of mitochondrial PT. oligodendrocyte mitochondria; 2',3'-cyclic nucleotide 3'-phosphodiesterase; permeability transition; calcium transport

Activation of the G protein [G.sub.s] results in increases in cAMP, a necessary step in the pathway for ATP release from rabbit and human erythrocytes. In all cells, the level of cAMP is the product of its synthesis by adenylyl cyclase and its hydrolysis by phosphodiesterases (PDEs). Both iloprost (I1o), a PG[I.sub.2] analog, and isoproterenol (Iso), a [beta]-agonist, stimulate receptor-mediated increases in cAMP in rabbit and human erythrocytes. However, the specific PDEs associated with each of these signaling pathways in the erythrocyte have not been fully characterized. Previously, we reported that PDE3B is present in rabbit and human erythrocyte membranes and that PDE3 inhibitors potentiate Ilo-induced increases in cAMP. Here we report that inhibitors of either PDE2 or PDE4, erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) and rolipram, respectively, potentiate Iso-induced increases in cAMP in rabbit and human erythrocytes. Importantly, these inhibitors had no effect on cAMP increases associated with the incubation of erythrocytes with Ilo. In addition, we establish, for the first time, the presence of PDE2A protein in rabbit and human erythrocyte membranes. Finally, we determined that preincubation of human erythrocytes with EHNA and rolipram together potentiate Iso-induced ATP release, whereas preincubation with cilostazol enhances Ilo-induced release of ATP. These results are consistent with the hypothesis that, in rabbit and human erythrocytes, Ilo-induced increases in cAMP and ATP release are regulated by PDE3, whereas those associated with Iso are regulated by the activities of both PDE2 and PDE4. These studies demonstrate that PDE activity in these cells is localized to specific signaling pathways. red blood cell; adenosine 3', 5'-cyclic monophosphate; adenosine 5'-trisphophate; rolipram: [beta]-adrenergic receptors

The gastric parietal cell is responsible for the secretion of HCl into the lumen of the stomach mainly due to stimulation by histamine via the cAMP pathway. However, the participation of several other receptors and pathways have been discovered to influence both stimulation and inhibition of acid secretion (e.g., cholinergic). Here we examine the role of phosphoinositide 3-kinase (PI3K) in the modulation of acid secretion. Treatment of isolated gastric glands and parietal cells with the PI3K inhibitor, LY294002 (LY), potentiated acid secretion in response to histamine to nearly the maximal secretion obtained with histamine plus phosphodiesterase inhibitors. As cAMP levels were elevated in response to histamine plus LY, but other means of elevating cAMP (e.g., forskolin, dbcAMP) were not influenced by LY, we posited that the effect might require activation of G-protein-coupled histamine [H.sub.2] receptors, possibly through the protein kinase B pathway (also known as Akt). Study of downstream effectors of PI3K showed that histaminergic stimulation increased Akt phosphorylation, which in turn was blocked by inhibition of PI3K. Expression studies showed that high expression of active Akt decreased acid secretion, whereas dominant-negative Akt increased acid secretion. Taken together, these data suggest stimulation with histamine increases the activity of PI3K leading to increased activity of Akt and decreased levels of cAMP in the parietal cell. parietal cell; Akt; protein kinase B; phosphodiesterase; H-K-ATPase

ATP-sensitive potassium ([K.sub.ATP]) channels couple cellular metabolic status to changes in membrane electrical properties. Caffeine (1,2,7-trimethylxanthine) has been shown to inhibit several ion channels; however, how caffeine regulates [K.sub.ATP] channels was not well understood. By performing single-channel recordings in the cell-attached configuration, we found that bath application of caffeine significantly enhanced the currents of Kir6.2/SUR1 channels, a neuronal/pancreatic [K.sub.ATP] channel isoform, expressed in transfected human embryonic kidney (HEK)293 cells in a concentration-dependent manner. Application of nonselective and selective phosphodiesterase (PDE) inhibitors led to significant enhancement of Kir6.2/SUR1 channel currents. Moreover, the stimulatory action of caffeine was significantly attenuated by KT5823, a specific PKG inhibitor, and, to a weaker extent, by BAPTA/AM, a membrane-permeable [Ca.sup.2+] chelator, but not by H-89, a selective PKA inhibitor. Furthermore, the stimulatory effect was completely abrogated when KT5823 and BAPTA/AM were co-applied with caffeine. In contrast, the activity of Kir6.2/SUR1 channels was decreased rather than increased by caffeine in cell-free inside-out patches, while tetrameric Kir6.2LRKR368/369/370/ 371AAAA channels were suppressed regardless of patch configurations. Caffeine also enhanced the single-channel currents of recombinant Kir6.2/SUR2B channels, a nonvascular smooth muscle [K.sub.ATP] channel isoform, although the increase was smaller. Moreover, bidirectional effects of caffeine were reproduced on the KATP channel present in the Cambridge rat insulinoma G1 (CRI-G1) cell line. Taken together, our data suggest that caffeine exerts dual regulation on the function of [K.sub.ATP], channels: an inhibitory regulation that acts directly on Kir6.2 or some closely associated regulatory protein(s), and a sulfonylurea receptor (SUR)-dependent stimulatory regulation that requires cGMP-PKG and intracellular [Ca.sup.2+]-dependent signaling. phosphodiesterase; protein kinase; calcium; single channel; patch clamp doi: 10.1152/ajpcell.00326.2006

Pulmonary hypertension (PHT) is associated with increased vascular resistance due to sustained contraction and enhanced proliferation of pulmonary arterial smooth muscle cells (PASMC); the abnormal tone and remodeling in the pulmonary vasculature may relate, at least in part, to decreased cyclic nucleotide levels. Cyclic nucleotide phosphodiesterases (PDEs), of which 11 families have been identified, catalyze the hydrolysis of cAMP and cGMP. We tested the hypothesis that PASMC isolated from patients with PHT, either idiopathic pulmonary arterial hypertension (IPAH) or secondary pulmonary hypertension (SPH), have increased expression and activity of PDE isoforms that reduce the responsiveness of agents that raise cellular cAMP. Realtime PCR and immunoblotting demonstrated that the expression of PDE1A, PDE1C, PDE3B, and PDE5A was enhanced in PASMC from both IPAH and SPH patients compared with control PASMC. Consistent with this enhanced expression of PDEs, agonist-stimulated cAMP levels were significantly reduced in IPAH and SPH PASMC unless a PDE inhibitor was present. The use of specific PDE inhibitors revealed that an increase in PDE1 and PDE3 activity largely accounted for reduced agonist-induced cAMP levels and increased proliferation in IPAH and SPH PASMC. Treatment with PDE1Ctargeted small interference RNA enhanced cAMP accumulation and inhibited cellular proliferation to a greater extent in PHT PASMC than controls. The results imply that an increase in PDE isoforms, in particular PDE1C, contributes to decreased cAMP and increased proliferation of PASMC in patients with PHT. PDE1 isoforms may provide novel targets for the treatment of both primary and secondary forms of the disease. pulmonary circulation and disease; signal transduction

Previous studies have shown that endothelial nitric oxide (NO) synthase (eNOS)-derived NO is an important signaling molecule in ischemia-reperfusion (I-R) injury. Deficiency of eNOS-derived NO has been shown to exacerbate injury in hepatic and myocardial models of I-R. We hypothesized that transgenic overexpression of eNOS (eNOS-TG) would reduce hepatic I-R injury. We subjected two strains of eNOS-TG mice to 45 min of hepatic ischemia and 5 h of reperfusion. Both strains were protected from hepatic I-R injury compared with wild-type littermates. Because the mechanism for this protection is still unclear, additional studies were performed by using inhibitors and activators of both soluble guanylyl cyclase (sGC) and heme oxygenase-1 (HO-1) enzymes. Blocking sGC with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and HO-1 with zinc (III) deuteroporphyrin IX-2,4-bisethyleneglycol (ZnDPBG) in wild-type mice increased hepatic I-R injury, whereas pharmacologically activating these enzymes significantly attenuated I-R injury in wild-type mice. Interestingly, ODQ abolished the protective effects of eNOS overexpression, whereas ZnDPBG had no effect. These results suggest that hepatic protection in eNOS-TG mice may be mediated in part by NO signaling via the sGC-cGMP pathway and is independent of HO-1 signal transduction pathways. nitric oxide; heine oxygenase-1; soluble guanyly cyclase; phosphodiesterase type 5 inhibition; endothelial nitric oxide synthase

There is a growing appreciation that the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling pathway is organized to form transduction units that function to deliver specific messages. Such organization results in the local activation of PKA subsets through the generation of confined intracellular gradients of cAMP, but the mechanisms responsible for limiting the diffusion of cAMP largely remain to be clarified. In this study, by performing real-time imaging of cAMP, we show that prostaglandin 1 stimulation generates multiple contiguous, intracellular domains with different cAMP concentration in human embryonic kidney 293 cells. By using pharmacological and genetic manipulation of phosphodiesterases (PDEs), we demonstrate that compartmentalized PDE4B and PDE4D are responsible for selectively modulating the concentration of cAMP in individual subcellular compartments. We propose a model whereby compartmentalized PDEs, rather than representing an enzymatic barrier to cAMP diffusion, act as a sink to drain the second messenger from discrete locations, resulting in multiple and simultaneous domains with different cAMP concentrations irrespective of their distance from the site of cAMP synthesis.

Phosphodiesterases (PDE) metabolize cyclic nucleotides limiting the effects of vasodilators such as prostacyclin and nitric oxide (NO). In this study, DNA microarray techniques were used to assess the impact of NO on expression of PDE genes in rat pulmonary arterial smooth muscle cells (rPASMC). Incubation of rPASMC with S-nitroso-L-glutathione (GSNO) increased expression of a PDE isoform that specifically metabolizes cAMP (PDE4B) in a dose- and time-dependent manner. GSNO increased PDE4B protein levels, and rolipram-inhibitable PDE activity was 2.3 [+ or -] 1.0-fold greater in GSNO-treated rPASMC than in untreated cells. The soluble guanylate cyclase (sGC) inhibitor, 1H[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one, and the cAMP-dependent protein kinase inhibitor, H89, prevented induction of PDE4B gene expression by GSNO, but the protein kinase G (PKG) inhibitors, Rp-8-pCPT-cGMPs and KT-5823, did not. Incubation of rPASMC with IL-1[beta] and tumor necrosis factor-[alpha] induced PDE4B gene expression, an effect that was inhibited by L-[N.sup.6]-(1-iminoethyl)lysine, an antagonist of NO synthase 2 (NOS2). The GSNO-induced increase in PDE4B mRNA levels was blocked by actinomycin D but augmented by cycloheximide. Infection of rPASMC with an adenovirus specifying a dominant negative cAMP response element binding protein (CREB) mutant inhibited the GSNO-induced increase of PDE4B gene expression. These results suggest that exposure of rPASMC to NO induces expression of PDE4B via a mechanism that requires cGMP synthesis by sGC but not PKG. The GSNO-induced increase of PDE4B gene expression is CREB dependent. These findings demonstrate that NO increases expression of a cAMP-specific PDE and provide evidence for a novel 'cross talk' mechanism between cGMP and cAMP signaling pathways. cross talk; S-nitroso-L-glutathione; adenosine 3',5'-cyclic monophosphate; guanosine 3',5'-cyclic monophosphate

Some of the major concepts related to cyclic nucleotide phosphodiesterase (PDE) function and regulation which include PDE integration into signaling complexes, the emerging association of mutant PDE alleles with inherited diseases and the role of PDEs in generating subcellular signaling compartments are reviewed.

cGMP serves as the main second messenger of nitric oxide (NO). Antifibrotic effects of enhancing renal cGMP levels have recently been documented in experimental acute anti-Thy-1 glomerulonephritis. The present study compares the effects of the cGMP production-increasing soluble guanylate cyclase (sGC) stimulator BAY 41-2272 with those of the cGMP degradation-limiting phosphodiesterase inhibitor pentoxifylline (PTX) in a progressive model of renal fibrosis. At 1 wk after induction of anti-Thy-1-induced chronic glomerulosclerosis (cGS), rats were randomly assigned to groups as follows: cGS, cGS + BAY 41-2272 (10 mg x kg body [wt.sup.-] x [day.sup.-1]), or cGS + PTX (50 mg x kg body [wt.sup.-1] x [day.sup.-1]). BAY 41-2272 and PTX reduced systolic blood pressure significantly. At 16 wk, tubulointerstitial expressions of sGC mRNA and NO-induced cGMP synthesis were increased in untreated cGS animals, whereas their glomerular activity was depressed compared with normal controls. Tuhulointerstitial and glomerular cGMP production in response to NO were significantly enhanced in animals treated with BAY 41-2272, but not in those treated with PTX. BAY 41-2272 administration resulted in marked reductions of glomemlar and tubulointerstitial histological matrix accumulation, expression of TGF-[beta]1 and fibronectin, macrophage infiltration, and cell proliferation as well as improved renal function. In contrast, only moderate and nonsignificant renoprotective changes were observed in the cGS + PTX group. In conclusion, increasing renal cGMP production through BAY 41-2272 significantly improved renal NO-cGMP signaling and limited progression in anti-Thy-l-induced chronic renal fibrosis, whereas inhibition of cGMP degradation by PTX was only moderately effective. The findings indicate that pharmacological enhancement of renal cGMP levels by sGC stimulation represents a novel and effective antifibrotic approach in progressive kidney disorders. BAY 41-2272; pentoxifylline; TGF-[beta]1; progression

LPS is known to modulate macrophage responses during sepsis, including cytokine release, phagocytosis, and proliferation. Although agents that elevate cAMP reverse LPS-induced macrophage functions, whether LPS itself modulates cAMP and whether LPS-induced decreases in proliferation are modulated via a cAMP-dependent pathway are not known. Murine macrophages (RAW264.7 cells) were treated with LPS in the presence or absence of inhibitors of prostaglandin signaling, protein kinases, CaM, [G.sub.i] proteins, and NF-[kappa]B translocation or transcription/translation. LPS effects on CaMKII phosphorylation and the expression of relevant adenylyl cyclase (AC) isoforms were measured. LPS caused a significant dose (5-10,000 ng/ml)- and time (1-8 h)-dependent increase in forskolin-stimulated AC activity that was abrogated by pretreatment with SN50 (an NF-[Kappa]B inhibitor), actinomycin D, or cycloheximide, indicating that the effect is mediated via NF-[Kappa]B-dependent transcription and new protein synthesis. Furthermore, LPS decreased the phosphorylation state of CaMKII, and pretreatment with a CaM antagonist attenuated the LPS-induced sensitization of AC. LPS, cAMP, or PKA activation each independently decreased macrophage proliferation. However, inhibition of NF-[Kappa]B had no effect on LPS-induced decreased proliferation, indicating that LPS-induced decreased macrophage proliferation can proceed via PKA-independent signaling pathways. Taken together, these findings indicate that LPS induces sensitization of AC activity by augmenting the stimulatory effect of CaM and attenuating the inhibitory effect of CaMKII on isoforms of AC that are CaMK sensitive. cell culture; prostaglandin; phosphodiesterase; nuclear factor-[Kappa]B; calcium/calmodulin-dependent kinase; calmodulin

We previously reported that in mesenteric arteries from streptozotocin (STZ)-induced diabetic rats that 1) endothelium-derived hyperpolarizing factor (EDHF)-type relaxation is impaired, possibly due to a reduced action of cAMP via increased phosphodiesterase 3 (PDE3) activity (Matsumoto T, Kobayashi T, and Kamata K. Am J Physiol Heart Circ Physiol 285: H283-H291, 2003) and that 2) PKA activity is decreased (Matsumoto T, Wakabayashi K, Kobayashi T, and Kamata K. Am J Physiol Heart Circ Physiol 287: H1064-H1071, 2004). Here we investigated whether chronic treatment with cilostazol, a PDE3 inhibitor, improves EDHF-type relaxation in mesenteric arteries isolated from STZ rats. We found that in such arteries 1) cilostazol treatment (2 wk) improved ACh-, A-23187-, and cyclopiazonic acid-induced EDHF-type relaxations; 2) the ACh-induced cAMP accumulation was transient and sustained in arteries from cilostazol-treated STZ rats; 3) the EDHF-type relaxation was significantly decreased by a PKA inhibitor in the cilostazol-treated group, but not in the cilostazol-untreated group; 4) cilostazol treatment improved both the relaxations induced by cAMP analogs and the PKA activity level; and 5) PKA catalytic subunit (Cat-[alpha]) protein was significantly decreased, but the regulatory subunit RII-[beta] was increased (and the latter effect was significantly decreased by cilostazol treatment). These results strongly suggest that cilostazol improves EDHF-type relaxations in STZ rats via an increase in cAMP and PKA signaling. adenosine; 3',5'-cyclic monophosphate; diabetes; phosphodiesterase; protein kinase A; streptozotocin

Nucleotide pyrophosphatase/phosphodiesterase (NPP)-type ectophosphodiesterases are found at the cell surface as type-I or type-II transmembrane proteins, but are also found extracellularly as secreted or shedded enzymes. They hydrolyze pyrophosphate or phosphodiester bonds in a variety of extracellular compounds including nucleotides, (lyso)phospholipids and choline phosphate esters. Despite their structurally related catalytic domain, each enzyme has well-defined substrate specificity. Catalysis by NPPs affects processes as diverse as cell proliferation and motility, angiogenesis, bone mineralization and digestion. In addition, there is emerging evidence for non-catalytic functions of NPPs in cell signaling. NPP-type ectophosphodiesterases are also implicated in the pathophysiology of cancer, insulin resistance and calcification diseases, and they hold great promise as easily accessible therapeutic targets.

Heme-regulated phosphodiesterase from Escherichia coli ([DOS.sub.Ec]) catalyzes the hydrolysis of cyclic AMP (cAMP) in vitro and is regulated by the redox state of the bound heme. Changes in the redox state result in alterations in the three-dimensional structure of the enzyme, which is then transmitted to the functional domain to switch catalysis on or off. Because [DOS.sub.Ec] was originally cloned from E. coli genomic DNA, it has not been known whether it is actually expressed in wild-type E. coli. In addition, the turnover number of [DOS.sub.Ec] using cAMP as a substrate is only 0.15 [min.sup.-1], which is relatively low for a physiologically relevant enzyme. In the present study, we demonstrated for the first time that the [DOS.sub.Ec] gene and protein are expressed in wild-type E. coli, especially under aerobic conditions. We also developed a [DOS.sub.Ec] gene knockout strain ([DELTA]dos). Interestingly, the knockout of dos caused excess accumulation of intracellular cAMP (26-fold higher than in the wild-type strain) under aerobic conditions, whereas accumulation of cAMP was not observed under anaerobic conditions. We also found differences in cell morphology and growth rate between the mutant cells and the wild-type strain. The changes in the knockout strain were partially complemented by introducing an expression plasmid for dos. Thus, the present study revealed that expression of DOSE, is regulated according to environmental [O.sub.2] availability at the transcriptional level and that the concentration of cAMP in cells is regulated by [DOS.sub.Ec] expression.

A study was conducted in which protein microarray system was used to examine the effects of the substrate, inhibitors, and various deletion and site-directed mutations on the protein-protein interactions associated with catalysis by Ec DOc, a heme-regulated phosphodiesterase from Escherichia coli. It was found that catalysis by Ec DOS correlates closely with the interaction between the full-length enzyme and the isolated PAS fragment.

The EAL domain (also known as domain of unknown function 2 or DUF2) is a ubiquitous signal transduction protein domain in the Bacteria. Its involvement in hydrolysis of the novel second messenger cyclic dimeric GMP (c-di-GMP) was demonstrated in vivo but not in vitro. The EAL domain-containing protein Dos from Escherichia coli was reported to hydrolyze cyclic AMP (cAMP), implying that EAL domains have different substrate specificities. To investigate the biochemical activity of EAL, the E. coli EAL domain-containing protein YahA and its individual EAL domain were overexpressed, purified, and characterized in vitro. Both full-length YahA and the EAL domain hydrolyzed c-di-GMP into linear dimeric GMP, providing the first biochemical evidence that the EAL domain is sufficient for phosphodiesterase activity. This activity was c-di-GMP specific, optimal at alkaline pH, dependent on [Mg.sup.2+] or [Mn.sup.2+], strongly inhibited by [Ca.sup.2+], and independent of protein oligomerization. Linear dimeric GMP was shown to be 5'pGpG. The EAL domain from Dos was overexpressed, purified, and found to function as a c-di-GMP-specific phosphodiesterase, not as a cAMP-specific phosphodiesterase, in contrast to previous reports. The EAL domains can hydrolyze 5'pGpG into GMP, however, very slowly, thus implying that this activity is irrelevant in vivo. Therefore, c-di-GMP is the exclusive substrate of EAL. Multiple-sequence alignment revealed two groups of EAL domains hypothesized to correspond to enzymatically active and inactive domains. The domains in the latter group have mutations in residues conserved in the active domains. The enzymatic inactivity of EAL domains may explain their coexistence with GGDEF domains in proteins possessing c-di-GMP synthase (diguanulate cyclase) activity.

A general mechanism that governs RNA polymerase (RNAP) movement and response to regulatory inputs like pauses, terminators, and elongation factors is described. It is showed that E. coli RNAP moves by a complex Brownian ratchet mechanism that acts prior to phosphodiester bond formation.

We investigated the effects of prostacyclin analogs and isoform-selective phosphodiesterase (PDE) inhibitors, alone and in combination, on pulmonary vascular remodeling in vitro and in vivo. Vascular smooth muscle cells (VSMC) isolated from pulmonary (proximal and distal) and systemic circulations demonstrated subtle variations in expression of PDE isoform mRNA. However, using biochemical assays, we found PDE3 and PDE4 isoforms to be responsible for the majority of cAMP hydrolysis in all VSMC. In growth assays, the prostacyclin analogs cicaprost and iloprost inhibited mitogen-induced proliferation of VSMC in a cAMP-dependent manner. In addition, isoform-selective antagonists of PDEs 1, 3, or 4 inhibited VSMC proliferation, an effect that synergized with the effect of prostacyclin analogs. The inhibitory effects were greater in cells isolated from pulmonary circulation. In an in situ perfused rat lung preparation, administration of prostacyclin analogs or the PDE inhibitors vinpocetine (PDEI), cilostamide (PDE3), or rolipram (PDE4), but not EHNA (PDE2), attenuated acute hypoxic vasoconstriction (HPV). Combinations of agents led to a greater reduction in HPV. Furthermore, during exposure to hypoxia for 13 days, Wistar rats were treated with iloprost, rolipram, cilostamide, or combinations of these agents. Compared with normoxic controls, hypoxic animals developed pulmonary hypertension and distal pulmonary artery muscularization. These parameters were attenuated by iloprost+cilostamide, iloprost+rolipram, and cilostamide+rolipram but were not significantly affected by single agents. Together, these findings provide a greater understanding of the role of cAMP PDEs in VSMC proliferation and provide rationale for combined use of prostacylcin analogs plus PDE3/4 inhibitors in treatment of pulmonary vascular remodeling. smooth muscle cells; cyclic nucleotides; heterogeneity

Environmental signals trigger changes in the bacterial cell surface, including changes in exopolysaccharides and proteinaceous appendages that ultimately favour bacterial persistence and proliferation. Such adaptations are regulated in diverse bacteria by proteins with GGDEF and EAL domains. These proteins are predicted to regulate cell surface adhesiveness by controlling the level of a second messenger, the cyclic dinucleotide c-di-GMP. Genetic evidence suggests that the GGDEF domain acts as a nucleotide cyclase for c-di-GMP synthesis while the EAL domain is a good candidate for the opposing activity, a phosphodiesterase for c-di-GMP degradation.

Lysophosphatidic acid (LPA) is generated from precursors by 'autotaxin', a once enigmatic exo-phosphodiesterase implicated in tumor cell motility, while exogenous phospholipases D can also produce Progress in the understanding of LPA bioactivity, signaling and synthesis is revie

Despite initial promise, the development of type 4 phosphodiesterase (PDE4) inhibitors as antidepressants has not advanced significantly. This is due to an incomplete understanding of the functional importance of PDE4 subtypes and high-affinity and low-affinity inhibitor-binding conformers. However, recent developments have rekindled interest in the therapeutic potential of PDE4 inhibitors. First, PDE4 has been shown to be involved in cAMP signaling pathways that are affected by antidepressants. Second, data obtained using mouse knockout lines indicate that PDE4D and PDE4B mediate antidepressant effects. Third, it appears that the interaction of inhibitors with the high-affinity binding conformer of PDE4 is particularly important for antidepressant efficacy. These developments highlight the difficulties of dissociating the actions of PDE4 inhibitors and provide a guide for future research.

Mechanisms of leptin secretion from white adipocytes. Am J Physiol Cell Physiol 283: C244-C250, 2002. First published March 6, 2002; 10.1152/ajpcell.00033.2002.--The mechanisms regulating leptin secretion were investigated in isolated rat white adipocytes. Insulin (1-100 nM) linearly stimulated leptin secretion from incubated adipocytes for at least 2 h. The adrenergic agonists norepinephrine, isoproterenol (two nonselective [beta]-agonists), or CL-316243 (potent [[beta].sub.3]) all inhibited insulin (10 nM)-stimulated leptin release. The inhibitory effects of norepinephrine and isoproterenol could be reversed not only by the nonselective antagonist propranolol but also by the selective antagonists ICI-89406 ([[beta].sub.1]) or ICI-118551 ([[beta].sub.2]), the [[beta].sub.2]-antagonist being less effective than the [[beta].sub.1]. Insulin-stimulated leptin secretion could also be inhibited by a series of agents increasing intracellular cAMP levels, such as lipolytic hormones (ACTH and thyrotropin-stimulating hormone), various nonhydrolyzable cAMP analogs, pertussis toxin, forskolin, methylxanthines (caffeine, theophylline, IBMX), and specific inhibitors of phosphodiesterase III (imazodan, milrinone, and amrinone). Significantly, antilipolytic agents other than insulin (adenosine, nicotinic acid, acipimox, and orthovanadate) did not mimic the acute stimulatory effects of insulin on leptin secretion under these conditions. We conclude that norepinephrine specifically inhibits insulin-stimulated leptin secretion not only via the low-affinity [[beta].sub.3]-adrenoceptors but also via the high-affinity [[beta].sub.1]/[[beta].sub.2]-adrenoceptors. Moreover, it is suggested that 1) activation of phosphodiesterase III by insulin represents an important metabolic step in stimulation of leptin secretion, and 2) lipolytic hormones competitively counterregulate the stimulatory effects of insulin by activating the adenylate cyclase system. lipolytic hormones; phosphodiesterases; [[beta].sub.1-], [[beta].sub.2-], and [[beta].sub.3]-adrenoceptors.

Results indicate that cGMP-phosphodiesterase activation is initiated by an interaction between transducin-alpha with the gamma-subunit of the phosphodiesterase. The interaction results in a bend in the alpha-helicl structure of the gamma-subunit C-terrminus, which allows trnsducin-alpha reach the catalytic site of phosphodiesterase.

Research has been conducted on the phosphodiesterase A1 protein of Acetobacter xylinum, a bacterial cellulose synthesis regulator. Results indicate that this protein contains heme and is regulated by reversible O (sub)2 binding to heme.

The His-607 in metal-binding motif A and His-643 in motif B of cGMP-specific phosphodiesterase are crucial for catalysis. All class I PDEs have 18 invariant residues in the catalytic site, but they all use different residues for metal binding.

The hypothesis that differences in enzyme regulation of cyclic adenylic acid (cAMP) synthesis and degradation uniquely establish an elevated content in pulmonary microvascular endothelial cells (PMVECs) was tested. This was because the mechanism that enables PMVECs to form a more restrictive barrier to macromolecular flux than pulmonary arterial endothelial cells was unknown. The results showed that suppressed calcium entry and low adenosine triphosphate-to-cAMP conversion intrinsincally influenced calcium sensitivity.

Phototransduction in vertebrate photoreceptor ROS requires the hydrolysis of cGMP by cyclic GMP phosphodiesterase (PDE). PDE regulatory subunit P gamma is ribosylated by adenosine diphosphate (ADP) in endogenous ADP-ribosyltransferase only if the Pgamma is free or complexed in amphibian rod photoreceptor membranes. Researchers believe that some PDE may not accept GTP/Talpha signals since Pgamma is ADP-ribosylated and its ability to interacts with T alpha is abolished.

Research was conducted to examine the ability of selective inhibitors of the phosphodiesterase isoenzymes to modulate mitogenic signaling in cultured human airway smooth muscle cells (HASM). Type 3-selective phosphodiesterase inhibitor siguazodan, type 4-selective phosphodiesterase inhibitor rolipram and the nonselective inhibitor 3-isobutyl-1-methylxanthine were analyzed for their effects on the proliferation of cultured HASM cells. Results demonstrate the ability of nonselective and isoform-selective phosphodiesterase inhibitors to modulate proliferative responses of HASMs in culture.

Gamma phosphodiesterase (PDE) subunit C68S, Y84C mutant binds with higher affinity to PDE compared to wild-type gamma PDE. However, the mutant is a significantly less potent inhibitor of PDE activity. The mutated gamma PDE protein appears to compete for only half of the total sites occupied by the wild-type gamma PDE subunit. The PDE molecules that have one site occupied by the gamma PDE mutant can still be fully inhibited by wild type gamma PDE. These findings indicate that the two gamma PDE sites on alpha,beta PDE are not identical and that only one of the two sites is catalytically active.

The expression of phosphodiesterase (PDE) activity and PDE genes in the A549 epithelial cell line and in primary human airway epithelial cells (HAECs) were studied. Both A549 cells and HAECs expressed PDE4 gene activity, with lesser PDE1, PDE3 and PDE5 activity. Inhibition of PDE4 and PDE3 reduced secretion by these cells. These suggested that epithelial PDE may be an important target for PDE4 inhibitors in the development of the control of asthmatic inflammation.

Oligodeoxyribonucleotides with (5'S)-5',8-cyclo-2'-deoxyadenosine {(5'S)-CyclodAdo} at certain positions were synthesized. The results showed that the phosphodiester linkages between the (5'S)-CyclodAdo and 2'-deoxyribonucleosides toward the enzymatic hydrolysis were highly stable. Moreover, the (5'S)-CyclodA-T base pair exhibited less stability compared to the A-T base pair.

Research was conducted to characterize a novel compound that binds and influences the states of purified recombinant human type 4 phosphodiesterase B2B (HSPDE4B2B) with high affinity. A treated glass fiber filter mat was utilized to filter the binding mixtures while protein concentrations were prepared both by a modified Bradford analysis and by quantitative amino acid analysis. Results showed that that the compound can interact with the isostates of PDE 4 observed in (R)-rolipram binding.

A study was conducted to examine the phosphorylation of the P-gamma subunit of cyclic GMP phosphodiesterase by cAMP-dependent protein kinase. Several oligonucleotides were utilized in site-directed mutagenesis while mutations were determined by double-stranded DNA sequencing. Results indicated that P-gamma phosphorylation by cAMP-dependent protein kinase supports the regulation of phototransduction in vertebrate rod photoreceptors.

A study was conducted to characterize the asymmetric synthesis of the phosphodiesterase inhibitor, CDP840, by Jacobsen epoxidation. The epoxidation controlled the absolute chemistry of the inhibitor through the Z triaryl olefin. Experimental results were analyzed based on the skewed side-on approach model advocated by Jacobsen. Results showed a highly selective hydride reduction of an epoxide supporting a retention of configuration.

A study was conducted to examine the absence of a kinetic isotope effect in hammerhead ribozyme phosphodiester cleavage supporting the nonexistence of a proton-transfer process in the rate-limiting step of the cleavage reaction. The kinetic and thermodynamic parameters of chimeric RNA/DNA ribozyme were evaluated against each other and with the corresponding values of an all-RNA ribozyme. Results indicated that the cleavage rates directly correlated with the concentration of active complexes relative to inactive complexes.

A study was conducted to analyze the solid-phase synthesis of oligodeoxyribonucleotides supporting a 3'-3' internucleoside phosphodiester linkage. Thermal denaturation analysis and gel retardation assays were carried out to determine the formation of intermolecular triplex complexes by alternate strand recognition. Results indicated that a marked sequence-dependent behavior in triplex formation by modified 16-mers.

The melting temperatures (Tm) and the association and dissociation of kinetic and thermodynamic parameters for triplexes formation were used in a guanidinium-linked thymidyl nucleoside d(Tg)4-T-azido polycation binding. Results reveal that triplestranded helices were provided by d(Tg)4-T-azido polycation that binds to short strand d(pA)x oligomers (x=5 - 8). The interaction was favored at physiologic ionic strength than the complexing of DNA by DNA or RNA.

The structure/calcium affinity relationships of cation chelating residues in calcium-binding sites III and IV were examined. The calmodulin mutants, F92W and F92W/D133, were prepared by site-specific cassette-mediated mutagenesis. The results showed that D/E single-site mutation lowers the calcium affinity of site IV by 2760-fold and that of site III by 24-fold.

[[Beta].sub.2]-Adrenoceptor agonists provide a mainstay in the treatment of asthma worldwide. However, despite their ability to provide symptomatic relief, chronic or repeated exposure to [[Beta].sub.2]-adrenoceptor agonists does not resolve asthmatic inflammation, because of the rapid development of tolerance by pro-inflammatory and immune cells of the lung. The prevailing belief is that tolerance to the so-called non-bronchodilator actions of [[Beta].sub.2]-adrenoceptor agonists is largely attributable to direct receptor desensitization mediated by G protein receptor-coupled kinases and/or cAMP-dependent protein kinase. Here, Mark Giembycz suggests another, largely ignored, explanation for [[Beta].sub.2]-adrenoceptor desensitization that is based on the accelerated degradation of cAMP by phosphodiesterase.